JP4834830B2 - Rail molecule fixing method and nano-transport device - Google Patents

Description

  The present invention relates to a rail molecule fixing method and a nano transport device.

Conventionally, in the field of nanotechnology, which is a technology in the range of several nanometers to several tens of nanometers, chemical energy can be converted into motion, and engineering is performed using biomolecular motors commonly found in the biological world. Research on technology to create a typical device is underway. Kinesin, a type of biomolecular motor, is known to move on microtubules, which are a type of rail molecule, along with the hydrolysis of ATP (adenosine triphosphate) (for example, Non-patent documents 1 to 8). In this case, the microtubule has a polarity, and kinesin moves from the minus end side to the plus end side of the microtubule, so that the microtubule polarity is maintained in a predetermined direction while maintaining the microtubule activity. It is important to be oriented, ie oriented.
Vale, RD, Reese, TS and sheetz, MP, "Identification of a novel force-generating protein, kinesin, involved in microtubule-based motility," Cell, vol. 42, pp. 39-50 (1985) Howard, J., Hudspeth, AJ and Vale, R., "Movement of microtubules by single kinesin molecules," Nature, vol. 342, pp. 154-159 (1989) Vale, RD, Funatsu, T., Pierce, DW, Romberg, L., Harada, Y. and Yanagida, T., "Direct observation of single kinesin molecules moving along microtubules," Nature, vol. 380, pp. 451- 453 (1996) Svoboda, K., Schmidt, C., Schnapp, B, and Block, S., "Direct observation of kinesin stepping by optical trapping interferometry," Nature, vol. 365, pp. 721-727 (1993) Nishiyama, M., Muto, E., Inoue, y., Yanagida, T. and Higuchi, H., "Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules," Nat. Cell Biol., Vol . 3, pp. 425-428 (2001) Meyhofer, E. and Howard, J., "The force generated by a single kinesin molecule against an elastic load," Proc. Natl. Acad. Sci. USA, vol. 92, pp. 574-578 (1995) Coppin, CM, Pierce, DW, Hsu, L. and Vale, RD, "The load dependence of kinesin's mechanical cycle," Proc. Natl. Acad. Sci. USA, vol. 94, pp. 8539-8544 (1997) Kojima, H., Muto, E., Higuchi, H. and Yanagida, T., "Mechanics of Single Kinesin Molecules Measured by Optical Trapping Nanometry," Biophys. J., vol. 73, pp. 2012-2022 (1997)

  However, since the conventional technique is such that the microtubules are oriented and fixed throughout the device, the reagent used for fixation must be completely removed from the device even after the microtubules are fixed. In this case, the next kinesin loses its activity. That is, it is deactivated. For this reason, repeated reagent replacement is required, which is extremely complicated.

  The present invention solves the above-mentioned conventional problems, moves a rail molecule by a biomolecule motor attached on a base material, deactivates the biomolecule motor by irradiating light of a predetermined wavelength, and rails It is intended to provide a rail molecule fixing method and a nano-transport device that can fix a molecule in a predetermined position in a predetermined direction easily and reliably without using a reagent by fixing the molecule. Objective.

Therefore, in the rail molecule immobilization method of the present invention, a method for immobilizing rail molecules having a polarity and moving the biomolecule motor in a direction corresponding to the polarity, which is coated with a solution containing the biomolecule motor. A biomolecule motor is attached on a base material, a rail molecule is introduced, and ATP is applied to move the rail molecule by the biomolecule motor, and the rail molecule is moved into an orientation channel formed on the base material. When the rail molecule reaches a predetermined position, the rail molecule is oriented and fixed in a predetermined direction by irradiating light of a predetermined wavelength to deactivate the biomolecule motor.

  In another rail molecule fixing method of the present invention, an alignment channel is formed on the substrate by joining a channel forming member on the substrate, and the rail molecule is formed from one end of the alignment channel. Into the alignment channel, and the rail molecules are fixed at predetermined positions in the alignment channel.

  In still another rail molecule fixing method of the present invention, the channel forming member is removed after the rail molecules are fixed.

  In still another rail molecule fixing method of the present invention, the rail molecule is a cytoskeletal fiber.

  In still another rail molecule fixing method of the present invention, the predetermined wavelength is 420 to 500 [nm].

  In still another rail molecule fixing method of the present invention, the light is irradiated for 60 seconds or more.

  In still another rail molecule fixing method of the present invention, a plurality of the rail molecules are fixed in parallel with each other and oriented in the same direction.

  In still another rail molecule fixing method of the present invention, the rail molecules are further fixed so as to form a curve or a polygonal line.

  The nano transport device of the present invention includes a rail molecule fixed by the rail molecule fixing method, and a biomolecule motor that moves on the rail molecule in a direction according to the polarity of the rail molecule. A motor conveys a conveyed product along the rail molecule.

  The rail molecule for the nano transport device of the present invention was fixed by the rail molecule fixing method.

According to the present invention, in the rail molecule immobilization method, a method is provided for immobilizing a rail molecule having a polarity and moving the biomolecule motor in a direction corresponding to the polarity, and coating with a solution containing the biomolecule motor. A biomolecule motor is attached on a base material, a rail molecule is introduced, and ATP is applied to move the rail molecule by the biomolecule motor, and the rail molecule is moved into an orientation channel formed on the base material. When the rail molecule reaches a predetermined position, the rail molecule is oriented and fixed in a predetermined direction by irradiating light of a predetermined wavelength to deactivate the biomolecule motor.

  In this case, the rail molecules can be fixed in a predetermined position in a predetermined direction easily and reliably without using a reagent. Therefore, the biomolecule motor that moves on the rail molecule can be moved in a predetermined direction.

  In another rail molecule fixing method, an orientation channel is formed on the substrate by bonding a channel forming member on the substrate, and the rail molecule is oriented from one end of the orientation channel. The rail molecules are entered into the alignment channel, and the rail molecules are fixed at predetermined positions in the alignment channel.

  In this case, since the rail molecule enters the alignment channel with the end having a predetermined polarity at the head, the rail molecule can be reliably aligned and fixed in a predetermined direction. Further, since the rail molecule can be fixed so as to have a shape corresponding to the shape of the orientation channel, the biomolecule motor moving on the rail molecule can be moved along a path of an arbitrary shape.

  According to the present invention, the nano transport device has a rail molecule fixed by the rail molecule fixing method, and a biomolecular motor that moves on the rail molecule in a direction according to the polarity of the rail molecule, The biomolecule motor conveys a conveyed product along the rail molecules.

  In this case, the conveyed product can be conveyed in a predetermined direction.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a diagram showing a method for manufacturing a nanochannel matrix for fixing a microtubule in an embodiment of the present invention, and FIG. 2 is a diagram showing a configuration of a nanochannel for fixing a microtubule in an embodiment of the present invention. It is. 1A to 1D, (2) shows the top surface of the substrate, and (1) shows a cross section taken along the line AB of (2).

  FIG. 1 shows a flow of manufacturing an alignment channel for aligning and fixing a microtubule 31 described later as a rail molecule in a predetermined direction, that is, a matrix of a nanochannel 29 described later. . In the present embodiment, a biomolecular motor that transports organelles and the like in living cells is used. The biomolecular motor is also called a motor protein (Motor Protein), binds to a polar cytoskeletal fiber, and moves in a certain direction along the cytoskeletal fiber. There are dozens of types of biomolecular motors in the cell, and the biomolecular motor in the present embodiment may be of any type, such as myosin, dynein, etc. However, since the inventor of the present invention conducted an experiment using the kinesin 32 described later as the biomolecular motor, here, a case where the biomolecule motor is the kinesin 32 will be described. In addition, the rail molecule for moving the biomolecular motor is a cytoskeletal fiber in the cell as described above, and may be, for example, an actin filament, but the inventor of the present invention Since the experiment was performed using the microtubule 31 as the rail molecule, here, the case where the rail molecule is the microtubule 31 will be described.

In the case of manufacturing the master block of the nanochannel 29, first, an electron beam resist 12 (for example, SAL601) is applied to the entire surface of the master substrate 11 made of Si, SiO ₂ or the like, and patterning is performed by electron beam lithography. It was. Thereby, the pattern of the electron beam resist 12 as shown in FIG. 1A was formed on the upper surface of the substrate 11. The pattern corresponds to the nanochannel 29 that fixes the microtubule 31.

  Subsequently, a UV (ultraviolet) resist 13 (for example, S1805) was applied to the entire surface of the substrate 11, and patterning was performed by photolithography using ultraviolet rays. Thereby, the pattern of the UV resist 13 as shown in FIG. 1B was formed on the upper surface of the substrate 11. This pattern corresponds to access channels 26a and 26b, which will be described later, to which the nanochannel 29 is connected.

  Subsequently, the peripheral portion 14 on the surface of the substrate 11 was etched by deep reactive ion etching (DRIE) using the electron beam resist 12 and the UV resist 13 as a mask. As a result, the peripheral portion 14 was etched by a depth a as shown in FIG. The depth a is about 4 [μm], and corresponds to the depth of the nanochannel 29 that fixes the microtubule 31.

  Subsequently, when the electron beam resist 12 and the UV resist 13 were removed from the surface of the substrate 11, as shown in FIG. 1 (d), a matrix having the convex portions 15 could be obtained. The convex portion 15 corresponds to a nanochannel 29 and access channels 26a and 26b described later. Then, a prepolymer of PDMS (Polydimethylsiloxane) was applied so as to cover the surface of the substrate 11, the prepolymer was cured, and then peeled off. Thereby, the PDMS film 21 as shown in FIG. 2A could be obtained.

  In the example shown in FIG. 2A, the nanochannel 29 and the access channels 26a and 26b are formed in a portion 24 surrounded by a circle on the lower surface of the PDMS film 21. Then, as shown by an arrow 23 in FIG. 2A, the PDMS film 21 was bonded so as to be in close contact with a glass plate 22 as a base material indicated as Coverslip. The glass plate 22 functions as a cover plate. As a result, a channel forming member as shown in FIG. 2B was obtained. Also, the portion indicated by the arrow 25 in FIG. 2 (a) is an injection hole for injecting a buffer made of physiological saline or the like into the nanochannel 29 and the access channels 26a and 26b. , Through holes for injection.

  FIG. 2B shows the portion 24 in an enlarged manner. In this case, as shown in FIG. 2B, access channels 26a and 26b indicated by arrows are formed between the central convex portion 28 and the peripheral convex portions 27 located on both sides of the central convex portion 28. Has been. In FIG. 2B, the access channels 26a and 26b are indicated as Main channels (A) and (B).

  A plurality of nanochannels 29 are formed in the central convex portion 28. In FIG. 2 (b), the nanochannel 29 is shown as Nanoscale sub-channel (s). The nanochannel 29 has both ends connected to the access channels 26a and 26b, respectively, and has the same depth as the access channels 26a and 26b. The depth is about 4 [μm] as described above.

  FIG. 2C is a photograph of a scanning electron microscope (SEM) in which a portion 24 surrounded by a circle on the lower surface of the PDMS film 21 is observed. Access channels 26a and 26b are observed above and below the photograph, and a nanochannel 29 is observed in the center. In this case, the length of the nanochannel 29 is 50 [μm], the width is 500 to 2000 [nm], and the height is 4 [μm].

  FIG. 2D shows the nanochannel 29 visualized using a fluorescent dye. After joining the PDMS film 21 so as to be in close contact with the glass plate 22, when the buffer is injected from the injection hole, the buffer is introduced into the nanochannel 29 by capillary action. FIG. 2D shows the nanochannel 29 visualized with a fluorescent dye using tetramethylrhodamine having a concentration of 10 [mM]. In FIG. 2D, the width of the narrow nanochannel 29 on the right side is 500 [nm], and the width of the thick nanochannel 29 on the left side is 750 [nm].

  Next, a method for fixing the microtubule 31 by irradiating light of a predetermined wavelength will be described.

  FIG. 3 is a schematic diagram showing microtubules and kinesin in the embodiment of the present invention, FIG. 4 is a diagram showing the relationship between the wavelength of irradiation light and the inactivation of kinesin in the embodiment of the present invention, and FIG. FIG. 6 is a photograph showing the relationship between the light irradiation time and the movement of the microtubule in the embodiment of the present invention, and FIG. 6 is a photograph showing the relationship between the light irradiation time and the movement of the microtubule in the embodiment of the present invention.

  In FIG. 3, 31 is a microtubule as a rail molecule, and 32 is a kinesin as a linear drive biomolecule motor that moves along the microtubule 31. The microtubule 31 is one of three types of cytoskeletal fibers, and is a cylinder having a diameter of 25 nm and a length of several tens of μm obtained by polymerizing tubulin as a monomer. It is a filament provided with a structure, that is, a fiber. Tubulin is a heterodimer in which two globular polypeptides, α-tubulin and β-tubulin, are tightly bound by noncovalent bonds.

  Further, the microtubule 31 has polarity, and one end (left end in FIG. 3) is a plus end, and the other end (right end in FIG. 3) is a minus end. The positive end and the negative end are identified by the rate at which tubulin, a subunit constituting the microtubule 31, is polymerized, and the rate at which polymerization is high is called the positive end. The end that slowly extends is called the minus end.

  On the other hand, kinesin 32 is a protein molecule having a head portion of about 10 nm and a total length of about 80 nm. And it has two spherical head parts and an elongated twisted coil part, and the head part alternately repeats the attachment and dissociation to the microtubules 31 so that the fibers can be handled with both hands. Move forward step by step. In this case, the motion of the kinesin 32 consists of 8 [nm] steps, and the maximum velocity is about 800 [nm / s]. As shown by the arrows in FIG. 3, the microtubule 31 is moved from the minus end side. Move toward the positive end. The generative force of kinesin 32 is 5 to 8 [pN].

  In the present embodiment, a method of fixing the microtubule 31 with the kinesin 32 by inactivating the kinesin 32 by irradiating with light of a predetermined wavelength is adopted. In this method, first, a large number of kinesins 32 are fixed to the surface of the substrate. When the microtubule 31 is introduced here and ATP is given, the microtubule 31 starts to move by the action of the kinesin 32 as a biomolecular motor. That is, since the kinesin 32 fixed on the surface of the base material moves on the microtubule 31, the microtubule 31 moves on the surface of the base material or the like. When the microtubule 31 reaches a desired position, light having a predetermined wavelength is irradiated. As a result, the kinesin 32 is deactivated and stops moving while the microtubule 31 is attached to the head portion of the kinesin 32. Therefore, the microtubule 31 stops. In this case, the head portion of the kinesin 32 is maintained attached to the microtubule 31, so that the microtubule 31 is fixed.

The inventor of the present invention has found through experiments that the predetermined wavelength is 420 to 500 [nm]. Specifically, as a result of experiments conducted by combining four types of optical filters, results as shown in FIG. 4A could be obtained. The four types of optical filters are the following (1) to (4).
(1) L42: UV cut filter for cutting light with a wavelength of 420 [nm] or less (2) Robon: Infrared light cut filter for cutting light with a wavelength of 800 [nm] or more (3) IF550: wavelength of 500 Bandpass filter that transmits light of ˜600 [nm] (4) Y50: a filter that cuts light having a wavelength of 500 [nm] or less FIG. 4B shows four types of (1) to (4) The relationship of the wavelength of the light which the optical filter permeate | transmits is shown. In FIG. 4B, the horizontal axis indicates the wavelength.

  In addition, in the experiment, the kinesin 32 is deactivated and the movement of the microtubule 31 is stopped by irradiating light from a mercury lamp that has passed through the combined optical filter during an in vitro gliding assay. Is. The gliding assay was performed in a flow cell produced using two cover glasses or a cover glass and a slide glass. In this case, if the tail side (upper side in FIG. 3) of kinesin 32 is fixed on the glass in the flow cell, the head side (lower side in FIG. 3) moves the microtubule 31 when ATP is hydrolyzed. When the movement of the kinesin 32 as a biomolecular motor is stopped by irradiating the light, the movement of the microtubule 31 is stopped.

  In the experiment, first, as shown in the left column of FIG. 4 (a), the light of a mercury lamp that passed through all four types of optical filters (1) to (4) was irradiated, and a minute amount before and after the irradiation. The movement of the tube 31 was observed. In this case, kinesin 32 as a biomolecular motor continued to move and did not stop. 4B shows that the wavelength of light irradiated through all four types of optical filters (1) to (4) is 500 to 600 [nm].

  Next, as shown in the middle column of FIG. 4A, the light of the mercury lamp that has passed through the two types of optical filters (1) and (2) is irradiated, and the movement of the microtubule 31 before and after the irradiation. Was observed. In this case, kinesin 32 as a biomolecular motor stopped moving. 4B shows that the wavelength of the light irradiated through the two types of optical filters (1) and (2) is 420 to 800 [nm].

  Finally, as shown in the right column of FIG. 4A, the light of the mercury lamp that has passed through the three types of optical filters (1), (2), and (4) is irradiated, and a minute amount before and after the irradiation. The movement of the tube 31 was observed. In this case, kinesin 32 as a biomolecular motor continued to move and did not stop. 4B shows that the wavelength of the light irradiated through the three types of optical filters (1), (2), and (4) is 500 to 800 [nm].

  As a result of such experiments, it was found that the predetermined wavelength of light for inactivating kinesin 32 is 420 to 500 [nm] as described above. Therefore, the inventor of the present invention uses the light of the mercury lamp that has passed through the two types of optical filters (1) and (2) as light having a wavelength of 420 to 500 [nm] in order to deactivate kinesin 32. It was decided to use.

  Subsequently, the inventor of the present invention has found through further experiments that the irradiation time of light necessary for inactivating kinesin 32 is 60 seconds or more. Specifically, in the same manner as in the experiment for finding the wavelength of light that deactivates kinesin 32 described above, by irradiation with light from a mercury lamp having a wavelength of 420 to 500 [nm] during the gliding assay, kinesin An experiment was conducted to deactivate 32 and stop the movement of the microtubule 31. In this case, during the gliding assay, the irradiation time of the light from the mercury lamp of 100 [W] was changed from 10 seconds to 90 seconds, and the movement of the microtubule 31 before and after the irradiation was observed.

  And the result of the experiment for finding the irradiation time of the light of a mercury lamp required in order to deactivate kinesin 32 is shown by FIG. FIG. 5 shows the relationship between the light irradiation time of the mercury lamp and the movement of the microtubule 31, where the horizontal axis represents the light irradiation time of the mercury lamp and the vertical axis represents the proportion of the microtube 31 moving. is there. In FIG. 5, ● indicates the state before irradiation, and ■ indicates the state after irradiation. From FIG. 5, it can be said that if the irradiation time of the light from the mercury lamp is 60 seconds or more, the kinesin 32 can be deactivated and the movement of all the microtubules 31 can be stopped. Therefore, the inventor of the present invention decided to irradiate the mercury lamp light having the above wavelength for 60 seconds or more in order to deactivate the kinesin 32.

  Moreover, the light of the mercury lamp of wavelength 420-500 [nm] was irradiated with the dark field microscope, and the movement of the microtubule 31 before and after irradiation could be observed. The result of this observation is shown in FIG. FIG. 6 is a dark-field micrograph showing the movement of the microtubule 31 during the gliding assay before and after irradiation. Here, FIGS. 6 (1) to (3) are before irradiation and show that the microtubule 31 is moving. After that, when the kinesin 32 was deactivated by irradiating light of a mercury lamp having the above wavelength for 40 seconds, as shown in FIGS. 6 (4) to (6), the movement of the microtubule 31 was stopped and fixed. It was done.

  In this way, by irradiating with light from a mercury lamp having a wavelength of 420 to 500 [nm], the movement of the microtubule 31 is stopped and fixed because the kinesin 32 fixed on the glass is deactivated. Because. However, if the deactivated kinesin 32 dissociates the bond with the microtubule 31, the microtubule 31 cannot be fixed. However, since the kinesin 32 maintains the bond with the microtubule 31 even if it is deactivated by irradiation with light having a wavelength of 420 to 500 [nm], the microtubule 31 may be fixed at the place irradiated with the light having the wavelength. I understood.

  And the method of fixing a microtubule by irradiating light with a wavelength of 420 to 500 [nm] has the following advantages. As shown in FIG. 3, since kinesin 32 moves on the microtubule 31 from the minus end side toward the plus end side, the tip of the microtubule 31 moving during the gliding assay becomes the minus end. Therefore, by fixing the moving microtubule 31 while being visualized by a dark field micrograph, the microtubule 31 can be fixed in a state where the polarity is clear. That is, it can be fixed in an oriented state. And if a substance is conveyed by the kinesin 32 which moves on the microtubule 31 fixed in the state in which the polarity is clear, the conveyance direction is clear.

  Moreover, the microtubule 31 can be locally fixed by irradiating light with a wavelength of 420 to 500 [nm]. For example, as in photolithography, a specific region is irradiated with light of the wavelength using a mask. Since the kinesin 32 is deactivated only in the portion irradiated with light, only the microtubule 31 in the specific region can be fixed.

  Thus, when the microtubule 31 as a rail molecule reaches a predetermined position, the microtubule 31 is oriented and fixed in a predetermined direction by irradiating the light of the wavelength to deactivate the kinesin 32 as a biomolecule. can do. Thereby, for example, the microtubule 31 can be fixed in a state where the polarity is clear only in the nanochannel 29 formed by the channel forming member as shown in FIG.

  Next, a method for fixing the microtubule 31 in the nanochannel 29 will be described.

  FIG. 7 is a view showing a method for fixing a microtubule in a nanochannel according to the embodiment of the present invention, FIG. 8 is a photograph showing a state in which the microtubule is introduced into the nanochannel according to the embodiment of the present invention, and FIG. FIG. 4 is a photograph showing a state of beads moving on a microtubule in a nanochannel according to an embodiment of the present invention.

  Here, a method of introducing and fixing the microtubule 31 in the nanochannel 29 in the test unit 35 as shown in FIG. 7 in an oriented state will be described. In this case, as shown in FIG. 2A, the unit 35 is configured by bonding a PDMS film 21 as a channel forming member to a glass plate 22 as a base material so as to be in close contact with each other. FIG. 7 is a plan view of the upper PDMS with the central convex portion 28 and the peripheral convex portion 27 remaining, with the glass plate 22 bonded to the upper surface of the channel forming member as shown in FIG. The state where the film 21 is removed is shown.

  Note that a plurality of nanochannels 29 as alignment channels are formed by central protrusions 28 that are part of the channel forming member bonded to the glass plate 22. Access channels 26a and 26b indicated by arrows are formed between the central convex portion 28 and the peripheral convex portions 27 located on both sides of the central convex portion 28. In FIG. 7, the access channels 26a and 26b are indicated as Main channels (A) and (B). The bottom surface of the nanochannel 29 is constituted by the top surface of the glass plate 22.

  Then, the channel in the unit 35, that is, the access channels 26 a and 26 b and the nanochannel 29 is filled with a solution containing kinesin 32, and the upper surface of the glass plate 22 is coated with kinesin 32. Subsequently, when the microtubule 31 is introduced from the access channel 26a, the microtubule 31 is captured by the kinesin 32 in the access channel 26a as shown in FIG.

  In FIG. 7, the microtubule 31 is indicated by a thick arrow, the tip of the arrow is a minus end, and the rear end is a plus end. Here, since the microtubule 31 is rigid and there is a difference between the width of the access channel 26a and the width of the nanochannel 29, the microtubule 31 is formed in the nanochannel 29 as shown in FIG. It does not enter and adheres to the upper surface of the glass plate 22 only in the access channel 26a.

  Subsequently, when ATP with a concentration of 1 [mM] is added from the access channel 26a, the kinesin 32 moves on the microtubule 31 along with the hydrolysis of ATP. Therefore, the microtubule 31 is transported by the kinesin 32 fixed to the upper surface of the glass plate 22 and probably enters the nanochannel 29 as shown in FIG. In this case, by changing the width of the access channel 26a and the nanochannel 29, the concentration of the microtubule 31, and the assay time, approximately one minute in one nanochannel 29 (for example, a width of 500 [nm]). Optimized for tube 31 to enter. As described above, since kinesin 32 moves on the microtubule 31 from the minus end side toward the plus end side, the microtubule 31 moves in the direction indicated by the arrow.

  Then, when the microtubule 31 enters the nanochannel 29, irradiation with light from a mercury lamp having a wavelength of 420 to 500 [nm] causes the kinesin 32 to be deactivated and the microtubule 31 is fixed. Therefore, as shown in FIG. 7 (3), the microtubule 31 can be fixed in a state in which the polarity is clearly aligned in the nanochannel 29. Thereby, the nano conveyance device fixed in the state where the microtubule 31 as the rail molecule is oriented can be obtained.

  Subsequently, as shown in FIG. 7 (4), when the kinesin beads 36 are introduced from the access channel 26b and ATP having a concentration of 1 [mM] is added, the kinesin beads 36 that have accessed the microtubule 31 are transferred to the access channel 26a. It is conveyed toward. Here, the kinesin beads 36 are polystyrene beads (diameter 320 [nm], Bangs Lab.) Coated with kinesin 32 on the surface, and can be obtained by mixing and incubating the polystyrene beads and kinesin 32. It was. In this case, when the kinesin 32 fixed to the kinesin bead 36 moves on the microtubule 31, the kinesin bead 36 as a transported object is transported from the minus end to the plus end of the microtubule 31. Become.

  The inventors of the present invention visualized the appearance of the microtubule 31 entering the nanochannel 29 having a width of 750 [nm] by using a fluorescent microtubule as the microtubule 31, as shown in FIG. . The fluorescent microtubules could be obtained by mixing and polymerizing fluorescent tubulin labeled with a fluorescent dye and tubulin not labeled with a fluorescent dye in an appropriate ratio. FIG. 8 is a series of photographs taken every 15 seconds and shows a state in which the microtubule 31 enters the nanochannel 29 by the gliding assay.

  In this case, as shown in FIG. 8 (1), after the minus end of the microtubule 31 entered the nanochannel 29, consecutive photographs were taken every 15 seconds until FIG. 8 (6). In addition, it seems that a part of the microtubule 31 is cut off in the middle, because the microtubule 31 is moved out of the focal depth (0.2 [μm]) of the objective lens used for photographing. This is because the kinesin 32 moves not only on the upper surface of the glass plate 22 but also on the side wall surface of the nanochannel 29 of the central convex portion 28 made of PDMS. This is because it moves up. FIG. 8 (7) shows a state in which the microtubule 31 has entered the plurality of nanochannels 29.

  In the example shown in FIG. 8, the nanochannel 29 visible in the center of the photograph has a length of 50 [μm], a width of 500 to 750 [nm], and a height (depth) of 4 [μm]. . Further, the access channels 26a and 26b as the main channels (A) and (B) visible on the left and right of the photograph have a width of 300 [μm] and a height (depth) of 4 [μm].

  Further, as shown in FIG. 9, the inventor of the present invention fixed the non-fluorescent microtubule 31 in an aligned state in the nanochannel 29, and then attached the kinesin beads to the access channel 26 b as the main channel (B). 36 was introduced, and a bead assay was performed on the microtubule 31 in the nanochannel 29. FIG. 9 (1) is a photograph showing a state in which the kinesin beads 36 are introduced into the nanochannel 29, and FIGS. 9 (2) to (4) are continuous photographs taken every 20 seconds. A state in which the kinesin beads 36 are conveyed through the nanochannel 29 is shown.

  In this case, the kinesin bead 36 adheres to one end (minus end) on the access channel 26b side of the microtubule 31, and then reaches 800 [nm] toward the access channel 26a side (the left side in FIG. 9) as the main channel (A). / S]. That is, it was confirmed that the kinesin beads 36 were transported in one direction at a transport speed of 800 [nm / s]. Thereby, it was shown that the microtubules 31 were fixed in the nanochannel 29 in an oriented state.

  Thus, in the present embodiment, microtubules as rail molecules in nanochannels 29 as alignment channels formed on glass plate 22 as a substrate coated with kinesin 32 as a biomolecular motor. The microtubule 31 is fixed in the nanochannel 29 by moving 31 and inactivating the kinesin 32 by irradiating light of a predetermined wavelength, that is, a wavelength of 420 to 500 [nm]. In this case, since the microtubule 31 enters the nanochannel 29 with the minus end as the head, it is oriented and fixed in a predetermined direction. Therefore, the microtubule 31 can be fixed in a predetermined direction in a predetermined direction easily and reliably without using a reagent. Therefore, the kinesin 32 moving on the microtubule 31 can be moved in a predetermined direction, that is, in the plus end direction.

  In the present embodiment, only the case where the shape of the nanochannel 29 is a straight line has been described. However, the shape of the nanochannel 29 may be a curved line, a broken line, or any shape. It may be. Further, the shape of the nanochannel 29 may be a three-dimensional curve or a broken line instead of a two-dimensional curve or a broken line. For example, when the surface of the glass plate 22 is not a flat surface but a curved surface, or when the surface of the glass plate 22 is uneven, the shape of the nanochannel 29 is a curve or a broken line that bends in a three-dimensional direction. Can do. The channel forming member for forming the nanochannel 29 is made of PDMS and has flexibility, so that it can be easily attached to the surface of the glass plate 22 having a curved surface or unevenness. Moreover, the length of the nanochannel 29 can also be set arbitrarily. Thereby, the path | route of the conveyed product conveyed by the kinesin 32 on the fixed microtubule 31 can be set so that it may pass arbitrary places with arbitrary length and arbitrary shapes.

  Further, the central convex portion 28 and the peripheral convex portion 27 as channel forming members for forming the nanochannel 29 can be removed from the glass plate 22 after the microtubule 31 is fixed. That is, since the kinesin 32 moves on the microtubule 31 and transports the transported object regardless of the presence or absence of the nanochannel 29, the transported object is fixed to the fixed microtubule 31 even if the nanochannel 29 is not present. Conveyed along.

  Then, the microtubule 31 fixed on the glass plate 22 by the above-described method and the kinesin 32 that moves on the microtubule 31 in the plus end direction transport a micro-transport object like the kinesin bead 36. Can be obtained. In this case, since the microtubules 31 as rail molecules are oriented and fixed in a predetermined direction, the transported object is transferred to the predetermined by the kinesin 32 as a biomolecule motor that moves in a direction corresponding to the polarity of the microtubules 31. Can be conveyed in the direction. In addition, as described above, since the microtubule 31 can be fixed in any length, in any shape, and through any location, the nano-transport device allows a transported object to have any length. Thus, it can be transported in a path that passes through an arbitrary place in an arbitrary shape. Furthermore, since the microtubules 31 can be fixed in a predetermined direction in a predetermined direction easily and reliably, the nano-transport device can be manufactured easily, reliably, and at low cost.

  Moreover, even if it is a large sized conveyance thing, it can be conveyed by fixing the microtubule 31 multiple pieces as a rail molecule | numerator so that it may mutually orientate in the same direction. That is, when there is one microtubule 31, for example, it can be transported if it is a transported object having a size of a rectangular parallelepiped having a length of 5 [μm], a width of 5 [μm], and a thickness of 2 [μm], Larger items cannot be conveyed. On the other hand, when a plurality of microtubules 31 are fixed in parallel with each other and oriented in the same direction, a plurality of kinesins 32 fixed to the conveyed product are identical on the plurality of microtubules 31. By moving in parallel in the direction, even a large transported object can be transported. In addition, even if a conveyed product is large sized, the several kinesin 32 can be fixed to the surface by immersing this conveyed product in a kinesin solution. Thereby, for example, a transported object having a size of a rectangular parallelepiped having a length of 5 [mm], a width of 5 [mm], and a thickness of 2 [μm] can be transported.

  In addition, this invention is not limited to the said embodiment, It can change variously based on the meaning of this invention, and does not exclude them from the scope of the present invention.

It is a figure which shows the method of manufacturing the matrix of the nanochannel which fixes a microtubule in embodiment of this invention. It is a figure which shows the structure of the nanochannel which fixes the microtubule in embodiment of this invention. It is a schematic diagram which shows the microtubule and kinesin in embodiment of this invention. It is a figure which shows the relationship between the wavelength of the irradiation light in the embodiment of this invention, and the deactivation of kinesin. It is a figure which shows the relationship between the irradiation time of the light and movement of a microtubule in embodiment of this invention. It is a photograph which shows the relationship between the irradiation time of the light and movement of a microtubule in embodiment of this invention. It is a figure which shows the method to fix the microtubule in embodiment of this invention in a nanochannel. It is a photograph which shows a mode that a microtubule introduce | transduces in the nanochannel in embodiment of this invention. It is a photograph which shows the mode of the bead which moves on the microtubule in the nanochannel in embodiment of this invention.

Explanation of symbols

21 PDMS film 22 Glass plate 27 Peripheral convex portion 28 Central convex portion 29 Nanochannel 31 Microtubule 32 Kinesin 36 Kinesin beads

Claims (10)

(A) A method of fixing a rail molecule having a polarity and moving a biomolecule motor in a direction corresponding to the polarity,
(B) attaching the biomolecule motor on the substrate by coating with a solution containing the biomolecule motor;
(C) by introducing a rail molecule and giving ATP, the biomolecule motor moves the rail molecule,
(D) The rail molecule enters the alignment channel formed on the substrate, and when the rail molecule reaches a predetermined position, the biomolecular motor is deactivated by irradiating with light of a predetermined wavelength. The rail molecule is fixed by aligning the rail molecules in a predetermined direction. (A) forming a channel for alignment on the substrate by bonding a channel forming member on the substrate;
(B) allowing the rail molecules to enter the alignment channel from one end of the alignment channel;
(C) The rail molecule fixing method according to claim 1, wherein the rail molecule is fixed at a predetermined position in the orientation channel. The rail molecule fixing method according to claim 2, wherein the channel forming member is removed after fixing the rail molecule. The rail molecule fixing method according to claim 1, wherein the rail molecule is a cytoskeleton fiber. The rail molecule fixing method according to claim 1, wherein the predetermined wavelength is 420 to 500 [nm]. The rail molecule fixing method according to claim 1, wherein the light is irradiated for 60 seconds or more. The rail molecule fixing method according to claim 1, wherein the plurality of rail molecules are fixed parallel to each other and oriented in the same direction. The rail molecule fixing method according to claim 1, wherein the rail molecule is fixed so as to form a curve or a broken line. (A) a rail molecule fixed by the rail molecule fixing method according to any one of claims 1 to 8, and
(B) a biomolecular motor that moves on the rail molecule in a direction corresponding to the polarity of the rail molecule;
(C) The nano-transport device, wherein the biomolecule motor transports a transport object along the rail molecule. A rail molecule for a nano transport device, which is fixed by the rail molecule fixing method according to any one of claims 1 to 8.

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